DNA molecule encoding a variant alpha2B-adrenoceptor protein, and uses thereof

ABSTRACT

This invention relates to a DNA sequence comprising a nucleotide sequence encoding a variant α 2B -adrenoceptor protein and to said variant α 2B -adrenoceptor protein as well as a method for screening a subject to determine if said subject is a carrier of a variant gene that encodes said variant α 2B -adrenoceptor. Further this invention relates to a method for treating a mammal suffering from vascular contraction of coronary arteries, said method comprising the step of administering a selective α 2B -adrenoceptor antagonist to said mammal and to transgenic animals comprising a human DNA molecule encoding human α 2B -adrenoceptor or said variant α 2B -adrenoceptor.

FIELD OF THE INVENTION

[0001] This invention relates to a DNA molecule encoding a variant humanα_(2B)-adrenoceptor, said variant α_(2B)-adrenoceptor protein and amethod to assess the risk of individuals to suffer from vascularcontraction of coronary arteries in mammals as well as a method for thetreatment of vascular contraction of coronary arteries. This inventionalso relates to transgenic animals comprising a human DNA moleculeencoding human α_(2B)-adrenoceptor or said variant α_(2B)-adrenoceptor.

BACKGROUND OF THE INVENTION

[0002] The publications and other materials used herein to illuminatethe background of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference.

[0003] The α₂-adrenoceptors α₂-ARs) mediate many of the physiologicaleffects of the catecholamines norepinephrine and epinephrine. Threegenetic subtypes of α₂-adrenoceptors are known in humans and othermammals, denoted as α_(2A)-, α_(2B)-, and α_(2C)-adrenoceptors. Thehuman genes encoding the receptors are located on chromosomes 10, 2 and4, respectively. No splice variants are known to exist of thesereceptors, as the genes are intronless. The tissue distributions andphysiological and pharmacological functions of the receptor subtypeshave been reviewed e.g. by MacDonald et al. (1997) and Docherty (1998).Based on recent studies with gene-targeted and transgenic mice,α_(2A)-adrenoceptors mediate most of the pharmacological actionsascribed to currently available α₂-adrenoceptor agonists, includinginhibition of neurotransmitter release, central hypotensive andbradycardic effects, sedation and anesthesia, and analgesia. The samestudies indicate that α_(2B)-adrenoceptors mediate peripheralvasoconstriction in response to agonist activation (Link et al. 1996,Macmillan et al. 1996). Other physiological or pharmacological effectshave not been associated with certainty with this receptor subtype. Theα_(2C)-adrenoceptor subtype appears to be involved in regulation ofcomplex behaviors. It is not known that this subtype would haveimportant functions in peripheral tissues outside the central nervoussystem or in cardiovascular regulation.

[0004] Coronary heart disease (CHD), like many other common disorders,arises from complex interactions between genetic and environmentalfactors. It is reasonable to assume that functionally important geneticvariation in mechanisms important for the regulation of vascularfunctions, including the coronary vasculature, will be found to beassociated with the pathogenesis and therapy of CHD. A variant form ofthe human α_(2B)-AR gene was recently identified (Heinonen et al.,1999). The variant allele encodes a receptor protein with a deletion ofthree glutamate residues in an acidic stretch of 18 amino acids (ofwhich 15 are glutamates) located in the third intracellular loop of thereceptor polypeptide. This acidic stretch is a unique feature in theprimary structure of α_(2B)-AR in comparison to α_(2A)-AR and α_(2C)-AR,suggesting that the motif has a distinct role in the function ofα_(2B)-AR. Amino acid sequence alignment of α_(2B)-AR polypeptides ofdifferent mammals reveals that the acidic stretch is highly conservedamong the α_(2B)-ARs of mammals and that the acidic stretch is long inhumans in comparison to other species. This suggests that the motif isimportant for the functionality of the receptor, and that the short form(D for “deletion”) probably represents the ancestral form and the longform (I for “insertion”) could well represent a more recent allelicvariant in humans. Jewell-Motz and Liggett (1995) studied the in vitrofunctions of this stretch using site-directed mutagenesis to delete aswell as to substitute 16 amino acids of the stretch. Their resultssuggest that this acidic motif is necessary for full agonist-promotedreceptor phosphorylation and desensitization.

[0005] Based on the vasoconstrictive property of α_(2B)-AR in mice andthe involvement of this acidic region in the desensitization mechanismof the receptor, we hypothesized that the deletion variant confersreduced receptor desensitization and therefore augmentedvasoconstriction that could be associated with cardiovascularpathologies. To test this hypothesis, we carried out a 4-yearprospective study in 912 middle-aged Finnish men.

OBJECT AND SUMMARY OF THE INVENTION

[0006] One object of this invention is to provide a DNA sequence of avariant human α_(2B)-adrenoceptor gene and the corresponding variantα_(2B)-adrenoceptor protein.

[0007] Another object of the invention is to provide a method forscreening a subject to assess if an individual is at risk to suffer fromvascular contraction of coronary arteries.

[0008] A third object of the invention is to provide a method for thetreatment of vascular contraction of coronary arteries of mammals.

[0009] A fourth object of the invention is to provide a transgenicanimal with a gene encoding a human α_(2B)-adrenoceptor or said variantthereof.

[0010] Thus, according to one aspect the invention concerns a DNAsequence comprising a nucleotide sequence encoding a variantα_(2B)-adrenoceptor protein with a deletion of at least 1 glutamate froma glutamic acid repeat element of 12 glutamates, amino acids 298-309, inan acidic stretch of 18 amino acids 294-311, located in the 3^(rd)intracellular loop of the receptor polypeptide.

[0011] The invention further concerns a variant α_(2B)-adrenoceptorprotein with a deletion of at least 1 glutamate from a glutamic acidrepeat element of 12 glutamates, amino acids 298-309, in an acidicstretch of 18 amino acids 294-311, located in the 3^(rd) intracellularloop of the receptor polypeptide.

[0012] According to another aspect the invention concerns a method forscreening a subject to determine if said subject is a carrier of a saidvariant gene with both alleles encoding a said variantα_(2B)-adrenoceptor, i.e. to determine if said subject's genotype of thehuman α_(2B)-adrenoceptor is of the deletion/deletion (D/D) type,comprising the steps of

[0013] a) providing a biological sample of the subject to be screened,

[0014] b) providing an assay for detecting in the biological sample thepresence of

[0015] i) the insertion/insertion (I/I) or deletion/insertion (D/I)genotypes of the human α_(2B)-adrenoceptor, or

[0016] ii) the D/D genotype of the human α_(2B)-adrenoceptor, and

[0017] c) assessing at least one of the two following

[0018] i) an individual's risk to develop a disease involving vascularcontraction of coronary arteries, or

[0019] ii) an individual's need for α_(2B)-selective orα_(2B)-nonselective α₂-adrenoceptor antagonist therapy,

[0020] based on whether said subject is of said D/D genotype or not.

[0021] According to a third aspect the present invention concerns amethod for treating a mammal suffering from vascular contraction ofcoronary arteries, said method comprising the step of administering aselective α_(2B)-adrenoceptor antagonist to said mammal.

[0022] According to a fourth aspect the present invention concerns atransgenic animal which carries a human DNA sequence comprising anucleotide sequence encoding a human α_(2B)-adrenoceptor protein or avariant thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention relates to a DNA molecule encoding avariant human α_(2B)-adrenoceptor, said variant α_(2B)-adrenoceptorprotein and a method to assess the risk of individuals to suffer fromvascular contraction of coronary arteries in mammals as well as a methodfor the treatment of vascular contraction of coronary arteries. Thepresent invention also relates to transgenic animals comprising a humanDNA molecule encoding a human α_(2B)-adrenoceptor or said variantα_(2B)-adrenoceptor protein.

[0024] The word treating shall also be understood to include preventing.

[0025] The concept “a deletion of at least 1 glutamate from a glutamicacid repeat element of 12 glutamates” refers to any deletion of 1 to 12glutamates irrespective of the specific location in, or how manyglutamates from said repeat element of 12 glutamates, amino acids298-309 (SEQ ID NO: 4), in an acidic stretch of 18 amino acids 294-311located in the 3^(rd) intracellular loop of the receptor polypeptide aredeleted.

[0026] The concept “deletion/deletion (D/D) genotype of the humanα_(2B)-adrenoceptor”, in short “D/D genotype”, refers to a genotype ofan individual having both α_(2B)-adrenoceptor alleles code for a variantα_(2B)-adrenoceptor with a deletion of at least 1 glutamate from aglutamic acid repeat element of 12 glutamates, amino acids 298-309, inan acidic stretch of 18 amino acids 294-311 (SEQ ID NO: 4), located inthe 3^(rd) intracellular loop of the receptor polypeptide.Correspondingly “deletion/insertion (D/I) genotype” refers to a genotypehaving one of the gene alleles code for an α_(2B)-adrenoceptor with asaid deletion and the other without a said deletion, i.e. with arespective insertion, and thus the “insertion/insertion (I/I) genotype”refers to a genotype having both alleles code for an α_(2B)-adrenoceptorwithout said deletion or deletions.

[0027] We recently identified a common variant form (SEQ ID NO: 1) ofthe human α_(2B)-AR gene (SEQ ID NO: 3). This variant gene encodes areceptor protein (SEQ ID NO: 2) with a deletion of 3 glutamates, aminoacids 307-309, from a glutamic acid (Glu) repeat element of 12glutamates, amino acids 298-309, in an acidic stretch of 18 amino acids294-311 (SEQ ID NO: 4), located in the 3^(rd) intracellular loop of thereceptor polypeptide. This variant gene (SEQ ID NO: 1) was associatedwith decreased basal metabolic rate (BMR) in a group of obese Finnishsubjects (Heinonen et al. 1999). Of the 166 obese subjects, 47 (28%)were homozygous for the long 12 glutamate repeat element (Glu¹²/Glu¹²),whereas 90 (54%) were heterozygous (Glu¹²/Glu⁹) and 29 (17%) werehomozygous for the short form (Glu⁹/Glu⁹).

[0028] The results to be presented below show that in a population-basedcohort of 912 Finnish middle-aged men subjects homozygous for the shortform (Glu⁹/Glu⁹) described above, thus representing a deletion/deletion(D/D) genotype of the α_(2B)-adrenoceptor, have a significantly elevatedrisk for acute coronary events in a four-year follow-up study. The riskfor an acute coronary event, defined as definite or possible acutemyocardial infarction (AMI) or prolonged (>20 min) chest pain requiringhospitalization, was increased 2.5 fold in subjects who had this D/Dgenotype. This increase in the risk for acute coronary events is asgreat as so far observed for any other genetic risk factor for acutecoronary events or acute myocardial infarction in a prospectivepopulation study. Also the frequency of a study subject having a historyof coronary heart disease (CHD) as well as CHD in an exercise test wasassociated with this D/D genotype. Based on these results and previouspublications referred to above it can be postulated that this D/Dgenotype is related to an impaired capacity to downregulateα_(2B)-adrenoceptor function during sustained receptor activation. Sincealtered α_(2B)-adrenoceptor function seems to be of relevance in thepathogenesis of a significant fraction of all cases of acute coronaryevents in subjects with this D/D genotype (homozygous Glu⁹/Glu⁹) webelieve it could also be of relevance in subjects with theinsertion/deletion (I/D) (heterozygous Glu¹²/Glu⁹) andinsertion/insertion (I/I) (homozygous Glu¹²/Glu¹²) genotypes when otherrisk factors for AMI are present. Further, since this specific deletionof 3 glutamates, amino acids 307-309, from said glutamic acid repeatelement of 12 glutamates, amino acids 298-309, in said acidic stretch of18 amino acids 294-311, located in the 3^(rd) intracellular loop of thereceptor polypeptide seems to be of relevance in cases of AMI we believethat also other deletions, i.e. deletions of at least 1 glutamate, fromsaid glutamic acid repeat element of 12 glutamates, amino acids 298-309,could be of relevance in the pathogenesis of AMI, because the 3^(rd)intracellular loop of the receptor polypeptide it is located in seems tohave an essential role in the downregulation of the α_(2B)-adrenoceptor.

[0029] Thus based on the results to be presented below and thepublications referred to above an α_(2B)-adrenoceptor antagonist wouldbe useful for treating a mammal suffering from vascular contraction ofcoronary arteries.

[0030] Furthermore, an α_(2B)-adrenoceptor antagonist selective for theα_(2B)-adrenoceptor subtype would be therapeutically beneficial for thetreatment of a disease involving said vascular contraction of coronaryarteries. Such a disease could be clinically expressed as chronic anginapectoris, specifically e.g. AMI, unstable angina pectoris orPrinzmetal's variant form of angina pectoris. If α_(2B)-adrenoceptordependent vasoconstriction is a causative factor in some cases of AMI,then antagonism of these receptors should restore coronary circulationand reduce the ischemic myocardial damage. An α_(2B)-adrenoceptorantagonist will relieve the vaso-constrictive component in the sustainedischemic episode of unstable angina pectoris, thus alleviating thesymptoms and preventing AMI. Vasoconstriction is a key factor in thepathogenesis of Prinzmetal's angina, and an α_(2B)-adrenoceptorantagonist may resolve and prevent attacks. An α_(2B)-adrenoceptorantagonist will help to alleviate the vasoconstrictive component in alltypes of CHD, providing both symptomatic relief and protection from AMI.

[0031] α_(2B)-adrenoceptors mediate vascular contraction of coronaryarteries, and genetic polymorphism present in the α_(2B)-adrenoceptorgene renders some subjects more susceptible to α_(2B)-adrenoceptormediated vasoconstriction of coronary arteries and associated clinicaldisorders. These subjects will especially benefit from treatment with anα_(2B)-adrenoceptor antagonist, and will be at increased risk foradverse effects if subtype-nonselective α₂-agonists are administered tothem. Therefore, a gene test recognizing subjects with a deletionvariant of the α_(2B)-adrenoceptor gene will be useful in diagnosticsand patient selection for specific therapeutic procedures. A gene testrecognizing the D/D genotype of the α_(2B)-adrenoceptor is useful inassessing an individual's risk to develop AMI and other clinicaldisorders involving vascular contraction of coronary arteries related tothe D/D genotype. A gene test recognizing the D/D genotype of theα_(2B)-adrenoceptor is useful in selecting drug therapy for patientswith diseases involving vascular contraction of coronary arteriesassociated with the D/D genotype; subjects with the D/D genotype willespecially benefit from therapy with α₂-adrenoceptor antagonistsα_(2B)-selective or nonselective). A gene test recognizing the D/Dgenotype of the α_(2B)-adrenoceptor is useful in selecting drug therapyfor patients who might be at increased risk for adverse effects ofα₂-adrenergic agonists; either, it will be possible to avoid the use ofα₂-agonists in such patients, or it will be possible to include aspecific α_(2B)-antagonist in their therapeutic regimen.

[0032] The DNA sequence can be used for screening a subject to determineif said subject is a carrier of a variant gene. The determination can becarried out either as a DNA analysis according to well known methods,which include direct DNA sequencing of the normal and variant gene,allele specific amplification using the polymerase chain reaction (PCR)enabling detection of either normal or variant sequence, or by indirectdetection of the normal or variant gene by various molecular biologymethods including e.g. PCR-single stranded conformation polymorphism(SSCP) method or denaturing gradient gel electrophoresis (DGGE).Determination of the normal or variant gene can also be done by using arestriction fragment length polymorphism (RFLP) method, which isparticularly suitable for genotyping large numbers of samples.Similarly, a test based on gene chip technology can be easily developedin analogy with many currently existing such tests for single-nucleotidepolymorphisms.

[0033] The determination can also be carried out at the level of RNA byanalyzing RNA expressed at tissue level using various methods. Allelespecific probes can be designed for hybridization. Hybridization can bedone e.g. using Northern blot, RNase protection assay or in situhybridization methods. RNA derived from the normal or variant gene canalso be analyzed by converting tissue RNA first to cDNA and thereafteramplifying cDNA by an allele specific PCR method.

[0034] As examples of useful α_(2B)-adrenoceptor antagonists can bementioned imiloxan [2-(1-ethyl-2-imidazoyl)methyl-1,4-benzodioxan,ARC-239[2-[2-(4-(2-methoxy-phenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione],prazosin[1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-furanylcarbonyl)piperazine]and chlorpromazine[2-chloro-N,N-dimethyl-10H-phenothiazine-10-propanamine].

[0035] The required dosage of the compounds will vary with theparticular condition being treated, the severity of the condition, theduration of the treatment, the administration route and the specificcompound being employed. A typical therapeutically effective daily doseadministered, e.g. orally or by infusion, can vary from e.g. 0.1 μg to10 mg per kilogram body weight of an adult person.

[0036] Influence of the variant gene sequence can be investigated intransgenic animals. A transgenic animal can be generated e.g. usingtargeted homologous recombination methodology. This will provide anideal preclinical model to investigate and screen new drug molecules,which are designed to modify the influence of the variant gene.

[0037] The invention will be described in more detail in theexperimental section.

EXPERIMENTAL SECTION

[0038] Determination of genomic alleles encoding the α_(2B)-adrenoceptor

[0039] PCR-SSCA analysis

[0040] The polymerase chain reaction-single stranded conformationalanalysis (PCR-SSCA) used to identify the genomic alleles encoding theα_(2B)-adrenoceptor was carried out as follows: The genomic DNA encodingthe α_(2B)-adrenergic receptor was amplified in two parts specific forthe intronless α_(2B)-adrenoceptor gene sequence (Lomasney et al. 1990).The PCR primer pairs for PCR amplification were as follows: Pair 1:5′-GGGGCGACGCTCTTGTCTA-3′ (SEQ ID NO: 5) and 5′-GGTCTCCCCCTCCTCCTTC-3′(SEQ ID NO: 6) (product size 878 bp), pair 2: 5′-GCAGCAACCGCAGAGGTC-3′(SEQ ID NO: 7) and 5′-GGGCAAGAAGCAGGGTGAC-3′ (SEQ ID NO: 8) (productsize 814 bp). The primers were delivered by KeboLab (Helsinki, Finland).PCR amplification was conducted in a 5 μl volume containing 100 nggenomic DNA (isolated from whole blood), 2.5 mmol/l of each primer, 1.0mmol/l deoxy-NTPs, 30 nmol/l ³³P-dCTP and 0.25 U AmpliTaq DNA polymerase(Perkin Elmer Cetus, Norwalk, Conn.). PCR conditions were optimizedusing the PCR Optimizer™ kit (Invitrogen, San Diego, Calif.). Sampleswere amplified with a GeneAmp PCR System 9600 (Perkin Elmer Cetus). PCRproducts were digested with restriction enzymes for SSCA analysis. Theproduct of primer pair 1 was digested with Dde I and Dra III (PromegaCorp., Madison, Wis.). The product of primer pair 2 was digested withAlu I and Hinc II (Promega Corp.). The digested samples were mixed withSSCA buffer containing 95% formamide, 10 mmol/l NaOH, 0.05% xylenecyanol and 0.05% bromophenol blue (total volume 25 μl). Before loading,the samples were denatured for 5 min at 95° C. and kept 5 min on ice.Three microliters of each sample were loaded on MDE™ high-resolution gel(FMC, BioProducts, Rockland, Mass.). The gel electrophoresis wasperformed twice, at two different running conditions: 6% MDE gel at +4°C. and 3% MDE gel at room temperature, both at 4 W constant power for 16h. The gels were dried and autoradiography was performed by apposing toKodak BioMax MR film for 24 h at room temperature.

[0041] Sequencing and genotyping

[0042] DNA samples migrating at different rates in SSCA were sequencedwith the Thermo Sequenase™ Cycle Sequencing Kit (Amersham Life Science,Cleveland, Ohio).

[0043] For genotyping the identified 3-glutamic acid deletion, DNA wasextracted from peripheral blood using standard methods. The α_(2B)-ARI/D genotype was determined by separating PCR-amplified DNA fragmentswith electrophoresis. Based on the nature of the I/D variant,identification of the long and short alleles was achieved by theirdifferent electrophoretic migration rates due to their 9 bp sizedifference.

[0044] The region of interest was amplified using a sense primer5′-AGGGTGTTTGTGGGGCATCT-3′ (SEQ ID NO: 9) and an anti-sense primer5′-CAAGCTGAGGCCGGAGACACT-3′ (SEQ ID NO: 10) (Oligold, Eurogentec,Belgium), yielding a product size of 112 bp for the long allele (I) and103 bp for the short allele (D). PCR amplification was conducted in a 10μL volume containing ˜100 ng genomic DNA, 1x buffer G (Invitrogen, SanDiego, Calif., USA), 0.8 mM dNTPs, 0.3 μM of each primer and 0.25 unitsof AmpliTaq DNA polymerase (Perkin Elmer Cetus, Norwalk, Conn., USA).Samples were amplified with a GeneAmp PCR System 9600 (Perkin ElmerCetus). After initial denaturation at 94° C. for 2 minutes, the sampleswere amplified over 35 cycles. PCR amplification conditions were 96° C.(40 s), 69° C. (30 s) and 72° C. (30 s) followed by final extension at72° C. for 6 minutes. The PCR products representing the long and shortalleles were identified by two alternative methods.

[0045] 1) The amplified samples were mixed with 4 μl of stop solution(Thermo Sequenase™ Cycle Sequencing kit), heated to 95° C. for 2 min,and loaded hot onto sequencing gels (Long Ranger™, FMC). The gels weredried and autoradiography was performed as previously described.

[0046] 2) Separation of the amplified PCR products was performed withelectrophoresis on a high-resolution 4% Metaphor agarose gel (FMCBioproducts, Rockland, Me.) and the bands were visualized by ethidiumbromide staining. In both methods, the long (Glu¹²) and short (Glu⁹)alleles were identified based on their different electrophoreticmigration rates.

[0047] Follow-up study

[0048] The above referred four-year follow-up study of 912 Finnishmiddle-aged men subjects including 192 subjects with a specificdeletion/deletion (D/D) genotype of the α_(2B)-adrenoceptor is describedin more detail in the following:

[0049] Knowing the vasoconstrictive property of α_(2B)-AR in mice andthe possible involvement of the investigated acidic region in thedesensitization mechanism of the receptor we hypothesized that theobserved insertion/deletion allelic variation could be associated withcardiovascular pathologies such as AMI. To test this hypothesis, wecarried out a four-year follow-up study in 912 middle-aged Finnish menwith no prior history of AMI. The study was carried out as part of theKuopio Ischemic Heart Disease Risk Factor Study (KIHD), which is anongoing population-based study designed to investigate risk factors forcardiovascular diseases and related outcomes in men from eastern Finland(Salonen 1988). This area is known for its homogenous population(Sajantila et al. 1996) and high coronary morbidity and mortality rates(Keys 1980).

[0050] Of the 912 subjects, 192 (21%) had the D/D genotype, 256 (28%)had the I/I genotype and 464 (51%) were heterozygous i.e. I/D. Thisgenotype distribution is in Hardy-Weinberg equilibrium (p=0.46).

[0051] Of the 37 cases that had an acute coronary event during thefollow-up, 18 were classified as definite AMI, 12 as possible AMI andseven as prolonged chest pain. Among the subjects with the D/D genotype,15 (8%) had an acute coronary event during the follow-up time. Thecorresponding incidences for the I/I and the heterozygous genotypes i.e.I/D were 10 (4%) and 12 (3%). The observed cumulative incidence of acutecoronary events differed significantly among the different genotypes(p=0.008). No significant difference in the cumulative incidence ofacute coronary events was found between the I/D and the I/I genotypes(p=0.4) (table 1). There was a significant difference (log-rankp=0.0045) between the D/D subgroup and the other two genotypes combinedin the cumulative event-free time in the Kaplan-Meier survival function,demonstrating that there is a consistently increased incidence of acutecoronary events in the D/D subgroup.

[0052] The D/D genotype was associated with a 2.5 fold increased riskfor an acute coronary event (95% CI=1.3-4.8, p=0.006) in comparison tothe other two genotypes combined. The relative risk remained above 2after adjustment for major CHD risk factors (table 2).

[0053] The D/D subgroup was not significantly different from the I/D+I/Isubgroup in terms of many known major risk factors for CHD. From 87variables in the study database only 5 were significantly differentbetween the D/D and the I/D+ I/I genotype subgroups: 1. there were moreacute coronary events in the D/D subgroup (8% vs. 3%, p=0.006), 2.history of CHD was more prevalent in the D/D subgroup (37% vs. 29%,p=0.043), 3. the prevalence of CHD in exercise test was higher in theD/D subgroup (30% vs. 22%, p=0.036), 4. mean hemoglobin level was higherin the D/D subgroup (149.0 g/l vs. 146.8 g/l, p=0.005) and 5. meandietary cholesterol intake (4-days) was lower in the D/D subgroup (411.6mg vs. 440.1 mg, p=0.033) (table 3). The first four observed differencessupport our hypothesis that the D/D genotype confers reduced receptordesensitization and therefore augmented vasoconstriction. This augmentedvasoconstriction is the reason for the increased incidence of acutecoronary events, the higher prevalence of CHD in exercise and history ofCHD. We hypothesize that the increased level of hemoglobin is due torelative anoxia of tissues because of this augmented vasoconstriction.

[0054] To examine the possibility that the D/D genotype is a geneticmarker for acute coronary events rather than a causative factor, we havesearched the literature for known genetic risk factors for acutecoronary events and AMI and their chromosomal localization. All but one(Apo-B) are on different chromosomes than the α_(2B)-AR gene (chromosome2) and the gene for Apo-B is neither in the physical nor the geneticvicinity of the α_(2B)-AR gene. Cox regression analysis revealed thatthe increased RR for acute coronary events in the D/D subgroup is notaffected by the serum Apo-B concentration.

[0055] Taken together, the known biological properties of the α_(2B)-AR,the homogeneity of the Finnish population with its relatively highincidence of CHD, the study design, the relatively large representativestudy population and the clustering of the findings around one traitsuggest that the D/D receptor allele is a causal genetic risk factor foracute coronary events. TABLE 1 The cumulative incidence of acutecoronary events among men with different genotypes of the α_(2B)-AR (pvalues are stated below) Genotype Events (% of men at risk) Men at risk(% of all) D/D observed 15 (8) 192 (21) expected  7.8 I/D observed 12(3) 464 (51) expected 18.8 I/I observed 10 (4) 256 (28) expected 10.4I/D + I/I observed 22 (3) 720 (79) expected 29.2 Total observed 37 (4) 912 (100)

[0056] P values for the above table:

[0057] D/D vs. I/D vs. I/I p=0.008

[0058] D/D vs. I/D p=0.002

[0059] D/D vs. I/I p=0.038

[0060] I/D vs. I/I p=0.389

[0061] D/D vs. I/D+I/I p=0.005 TABLE 2 Relative risk (RR) and its 95%confidence interval (CI) for an acute coronary event - a comparison ofeach of the genotypes with the other two combined. Results of a Coxregression model for 37 acute coronary events in a population sample of912 subjects Adjusted RR RR (95% CI) (95% CI) Genotype Events/men atrisk p p D/D 15/192  2.5(1.3-4.8) 2.3(1.2-4.5) 0.006 0.014 I/D 12/4640.44(0.2-0.9) 0.5(0.2-1.0) 0.020 0.052 I/I 10/256 1.03(0.5-2.1)0.96(0.5-2.0)  0.940 0.901

[0062] Adjustment was done for age, CHD in the family, high cholesterolin the family, hypertension and smoking TABLE 3 List of all significantdifferences (p < 0.05) between the D/D and the I/D + I/I genotypesubgroups among 87 variables in the study database Variable D/D I/D +I/I p Acute coronary events [event/n (%)] 15/192 (8)  22/720 (3) 0.006Ischemic findings in exercise test 57/192 (30) 160/720 (22) 0.036[case/n (%)] History of CHD [case/n (%)] 71/192 (37) 209/720 (29) 0.043Mean blood haemoglobin [g/L] 149.0 146.8 0.005 Mean 4 day dietarycholesterol intake 411.6 440.1 0.033 [mg]

[0063] %=Percent of men at risk

[0064] It will be appreciated that the methods of the present inventioncan be incorporated in the form of a variety of embodiments, only a fewof which are disclosed herein. It will be apparent for the specialist inthe field that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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1 10 1 1344 DNA Homo sapiens CDS (1)..(1341) Coding sequence for varianthuman alpha-2B-adrenoceptor protein 1 atg gac cac cag gac ccc tac tccgtg cag gcc aca gcg gcc ata gcg 48 Met Asp His Gln Asp Pro Tyr Ser ValGln Ala Thr Ala Ala Ile Ala 1 5 10 15 gcg gcc atc acc ttc ctc att ctcttt acc atc ttc ggc aac gct ctg 96 Ala Ala Ile Thr Phe Leu Ile Leu PheThr Ile Phe Gly Asn Ala Leu 20 25 30 gtc atc ctg gct gtg ttg acc agc cgctcg ctg cgc gcc cct cag aac 144 Val Ile Leu Ala Val Leu Thr Ser Arg SerLeu Arg Ala Pro Gln Asn 35 40 45 ctg ttc ctg gtg tcg ctg gcc gcc gcc gacatc ctg gtg gcc acg ctc 192 Leu Phe Leu Val Ser Leu Ala Ala Ala Asp IleLeu Val Ala Thr Leu 50 55 60 atc atc cct ttc tcg ctg gcc aac gag ctg ctgggc tac tgg tac ttc 240 Ile Ile Pro Phe Ser Leu Ala Asn Glu Leu Leu GlyTyr Trp Tyr Phe 65 70 75 80 cgg cgc acg tgg tgc gag gtg tac ctg gcg ctcgac gtg ctc ttc tgc 288 Arg Arg Thr Trp Cys Glu Val Tyr Leu Ala Leu AspVal Leu Phe Cys 85 90 95 acc tcg tcc atc gtg cac ctg tgc gcc atc agc ctggac cgc tac tgg 336 Thr Ser Ser Ile Val His Leu Cys Ala Ile Ser Leu AspArg Tyr Trp 100 105 110 gcc gtg agc cgc gcg ctg gag tac aac tcc aag cgcacc ccg cgc cgc 384 Ala Val Ser Arg Ala Leu Glu Tyr Asn Ser Lys Arg ThrPro Arg Arg 115 120 125 atc aag tgc atc atc ctc act gtg tgg ctc atc gccgcc gtc atc tcg 432 Ile Lys Cys Ile Ile Leu Thr Val Trp Leu Ile Ala AlaVal Ile Ser 130 135 140 ctg ccg ccc ctc atc tac aag ggc gac cag ggc ccccag ccg cgc ggg 480 Leu Pro Pro Leu Ile Tyr Lys Gly Asp Gln Gly Pro GlnPro Arg Gly 145 150 155 160 cgc ccc cag tgc aag ctc aac cag gag gcc tggtac atc ctg gcc tcc 528 Arg Pro Gln Cys Lys Leu Asn Gln Glu Ala Trp TyrIle Leu Ala Ser 165 170 175 agc atc gga tct ttc ttt gct cct tgc ctc atcatg atc ctt gtc tac 576 Ser Ile Gly Ser Phe Phe Ala Pro Cys Leu Ile MetIle Leu Val Tyr 180 185 190 ctg cgc atc tac ctg atc gcc aaa cgc agc aaccgc aga ggt ccc agg 624 Leu Arg Ile Tyr Leu Ile Ala Lys Arg Ser Asn ArgArg Gly Pro Arg 195 200 205 gcc aag ggg ggg cct ggg cag ggt gag tcc aagcag ccc cga ccc gac 672 Ala Lys Gly Gly Pro Gly Gln Gly Glu Ser Lys GlnPro Arg Pro Asp 210 215 220 cat ggt ggg gct ttg gcc tca gcc aaa ctg ccagcc ctg gcc tct gtg 720 His Gly Gly Ala Leu Ala Ser Ala Lys Leu Pro AlaLeu Ala Ser Val 225 230 235 240 gct tct gcc aga gag gtc aac gga cac tcgaag tcc act ggg gag aag 768 Ala Ser Ala Arg Glu Val Asn Gly His Ser LysSer Thr Gly Glu Lys 245 250 255 gag gag ggg gag acc cct gaa gat act gggacc cgg gcc ttg cca ccc 816 Glu Glu Gly Glu Thr Pro Glu Asp Thr Gly ThrArg Ala Leu Pro Pro 260 265 270 agt tgg gct gcc ctt ccc aac tca ggc cagggc cag aag gag ggt gtt 864 Ser Trp Ala Ala Leu Pro Asn Ser Gly Gln GlyGln Lys Glu Gly Val 275 280 285 tgt ggg gca tct cca gag gat gaa gct gaagag gag gaa gag gag gag 912 Cys Gly Ala Ser Pro Glu Asp Glu Ala Glu GluGlu Glu Glu Glu Glu 290 295 300 gag gag tgt gaa ccc cag gca gtg cca gtgtct ccg gcc tca gct tgc 960 Glu Glu Cys Glu Pro Gln Ala Val Pro Val SerPro Ala Ser Ala Cys 305 310 315 320 agc ccc ccg ctg cag cag cca cag ggctcc cgg gtg ctg gcc acc cta 1008 Ser Pro Pro Leu Gln Gln Pro Gln Gly SerArg Val Leu Ala Thr Leu 325 330 335 cgt ggc cag gtg ctc ctg ggc agg ggcgtg ggt gct ata ggt ggg cag 1056 Arg Gly Gln Val Leu Leu Gly Arg Gly ValGly Ala Ile Gly Gly Gln 340 345 350 tgg tgg cgt cga cgg gcg cag ctg acccgg gag aag cgc ttc acc ttc 1104 Trp Trp Arg Arg Arg Ala Gln Leu Thr ArgGlu Lys Arg Phe Thr Phe 355 360 365 gtg ctg gct gtg gtc att ggc gtt tttgtg ctc tgc tgg ttc ccc ttc 1152 Val Leu Ala Val Val Ile Gly Val Phe ValLeu Cys Trp Phe Pro Phe 370 375 380 ttc ttc agc tac agc ctg ggc gcc atctgc ccg aag cac tgc aag gtg 1200 Phe Phe Ser Tyr Ser Leu Gly Ala Ile CysPro Lys His Cys Lys Val 385 390 395 400 ccc cat ggc ctc ttc cag ttc ttcttc tgg atc ggc tac tgc aac agc 1248 Pro His Gly Leu Phe Gln Phe Phe PheTrp Ile Gly Tyr Cys Asn Ser 405 410 415 tca ctg aac cct gtt atc tac accatc ttc aac cag gac ttc cgc cgt 1296 Ser Leu Asn Pro Val Ile Tyr Thr IlePhe Asn Gln Asp Phe Arg Arg 420 425 430 gcc ttc cgg agg atc ctg tgc cgcccg tgg acc cag acg gcc tgg tga 1344 Ala Phe Arg Arg Ile Leu Cys Arg ProTrp Thr Gln Thr Ala Trp 435 440 445 2 447 PRT Homo sapiens 2 Met Asp HisGln Asp Pro Tyr Ser Val Gln Ala Thr Ala Ala Ile Ala 1 5 10 15 Ala AlaIle Thr Phe Leu Ile Leu Phe Thr Ile Phe Gly Asn Ala Leu 20 25 30 Val IleLeu Ala Val Leu Thr Ser Arg Ser Leu Arg Ala Pro Gln Asn 35 40 45 Leu PheLeu Val Ser Leu Ala Ala Ala Asp Ile Leu Val Ala Thr Leu 50 55 60 Ile IlePro Phe Ser Leu Ala Asn Glu Leu Leu Gly Tyr Trp Tyr Phe 65 70 75 80 ArgArg Thr Trp Cys Glu Val Tyr Leu Ala Leu Asp Val Leu Phe Cys 85 90 95 ThrSer Ser Ile Val His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Trp 100 105 110Ala Val Ser Arg Ala Leu Glu Tyr Asn Ser Lys Arg Thr Pro Arg Arg 115 120125 Ile Lys Cys Ile Ile Leu Thr Val Trp Leu Ile Ala Ala Val Ile Ser 130135 140 Leu Pro Pro Leu Ile Tyr Lys Gly Asp Gln Gly Pro Gln Pro Arg Gly145 150 155 160 Arg Pro Gln Cys Lys Leu Asn Gln Glu Ala Trp Tyr Ile LeuAla Ser 165 170 175 Ser Ile Gly Ser Phe Phe Ala Pro Cys Leu Ile Met IleLeu Val Tyr 180 185 190 Leu Arg Ile Tyr Leu Ile Ala Lys Arg Ser Asn ArgArg Gly Pro Arg 195 200 205 Ala Lys Gly Gly Pro Gly Gln Gly Glu Ser LysGln Pro Arg Pro Asp 210 215 220 His Gly Gly Ala Leu Ala Ser Ala Lys LeuPro Ala Leu Ala Ser Val 225 230 235 240 Ala Ser Ala Arg Glu Val Asn GlyHis Ser Lys Ser Thr Gly Glu Lys 245 250 255 Glu Glu Gly Glu Thr Pro GluAsp Thr Gly Thr Arg Ala Leu Pro Pro 260 265 270 Ser Trp Ala Ala Leu ProAsn Ser Gly Gln Gly Gln Lys Glu Gly Val 275 280 285 Cys Gly Ala Ser ProGlu Asp Glu Ala Glu Glu Glu Glu Glu Glu Glu 290 295 300 Glu Glu Cys GluPro Gln Ala Val Pro Val Ser Pro Ala Ser Ala Cys 305 310 315 320 Ser ProPro Leu Gln Gln Pro Gln Gly Ser Arg Val Leu Ala Thr Leu 325 330 335 ArgGly Gln Val Leu Leu Gly Arg Gly Val Gly Ala Ile Gly Gly Gln 340 345 350Trp Trp Arg Arg Arg Ala Gln Leu Thr Arg Glu Lys Arg Phe Thr Phe 355 360365 Val Leu Ala Val Val Ile Gly Val Phe Val Leu Cys Trp Phe Pro Phe 370375 380 Phe Phe Ser Tyr Ser Leu Gly Ala Ile Cys Pro Lys His Cys Lys Val385 390 395 400 Pro His Gly Leu Phe Gln Phe Phe Phe Trp Ile Gly Tyr CysAsn Ser 405 410 415 Ser Leu Asn Pro Val Ile Tyr Thr Ile Phe Asn Gln AspPhe Arg Arg 420 425 430 Ala Phe Arg Arg Ile Leu Cys Arg Pro Trp Thr GlnThr Ala Trp 435 440 445 3 1353 DNA Homo sapiens CDS (1)..(1350) Codingsequence for human alpha-2B-adrenoceptor protein 3 atg gac cac cag gacccc tac tcc gtg cag gcc aca gcg gcc ata gcg 48 Met Asp His Gln Asp ProTyr Ser Val Gln Ala Thr Ala Ala Ile Ala 1 5 10 15 gcg gcc atc acc ttcctc att ctc ttt acc atc ttc ggc aac gct ctg 96 Ala Ala Ile Thr Phe LeuIle Leu Phe Thr Ile Phe Gly Asn Ala Leu 20 25 30 gtc atc ctg gct gtg ttgacc agc cgc tcg ctg cgc gcc cct cag aac 144 Val Ile Leu Ala Val Leu ThrSer Arg Ser Leu Arg Ala Pro Gln Asn 35 40 45 ctg ttc ctg gtg tcg ctg gccgcc gcc gac atc ctg gtg gcc acg ctc 192 Leu Phe Leu Val Ser Leu Ala AlaAla Asp Ile Leu Val Ala Thr Leu 50 55 60 atc atc cct ttc tcg ctg gcc aacgag ctg ctg ggc tac tgg tac ttc 240 Ile Ile Pro Phe Ser Leu Ala Asn GluLeu Leu Gly Tyr Trp Tyr Phe 65 70 75 80 cgg cgc acg tgg tgc gag gtg tacctg gcg ctc gac gtg ctc ttc tgc 288 Arg Arg Thr Trp Cys Glu Val Tyr LeuAla Leu Asp Val Leu Phe Cys 85 90 95 acc tcg tcc atc gtg cac ctg tgc gccatc agc ctg gac cgc tac tgg 336 Thr Ser Ser Ile Val His Leu Cys Ala IleSer Leu Asp Arg Tyr Trp 100 105 110 gcc gtg agc cgc gcg ctg gag tac aactcc aag cgc acc ccg cgc cgc 384 Ala Val Ser Arg Ala Leu Glu Tyr Asn SerLys Arg Thr Pro Arg Arg 115 120 125 atc aag tgc atc atc ctc act gtg tggctc atc gcc gcc gtc atc tcg 432 Ile Lys Cys Ile Ile Leu Thr Val Trp LeuIle Ala Ala Val Ile Ser 130 135 140 ctg ccg ccc ctc atc tac aag ggc gaccag ggc ccc cag ccg cgc ggg 480 Leu Pro Pro Leu Ile Tyr Lys Gly Asp GlnGly Pro Gln Pro Arg Gly 145 150 155 160 cgc ccc cag tgc aag ctc aac caggag gcc tgg tac atc ctg gcc tcc 528 Arg Pro Gln Cys Lys Leu Asn Gln GluAla Trp Tyr Ile Leu Ala Ser 165 170 175 agc atc gga tct ttc ttt gct ccttgc ctc atc atg atc ctt gtc tac 576 Ser Ile Gly Ser Phe Phe Ala Pro CysLeu Ile Met Ile Leu Val Tyr 180 185 190 ctg cgc atc tac ctg atc gcc aaacgc agc aac cgc aga ggt ccc agg 624 Leu Arg Ile Tyr Leu Ile Ala Lys ArgSer Asn Arg Arg Gly Pro Arg 195 200 205 gcc aag ggg ggg cct ggg cag ggtgag tcc aag cag ccc cga ccc gac 672 Ala Lys Gly Gly Pro Gly Gln Gly GluSer Lys Gln Pro Arg Pro Asp 210 215 220 cat ggt ggg gct ttg gcc tca gccaaa ctg cca gcc ctg gcc tct gtg 720 His Gly Gly Ala Leu Ala Ser Ala LysLeu Pro Ala Leu Ala Ser Val 225 230 235 240 gct tct gcc aga gag gtc aacgga cac tcg aag tcc act ggg gag aag 768 Ala Ser Ala Arg Glu Val Asn GlyHis Ser Lys Ser Thr Gly Glu Lys 245 250 255 gag gag ggg gag acc cct gaagat act ggg acc cgg gcc ttg cca ccc 816 Glu Glu Gly Glu Thr Pro Glu AspThr Gly Thr Arg Ala Leu Pro Pro 260 265 270 agt tgg gct gcc ctt ccc aactca ggc cag ggc cag aag gag ggt gtt 864 Ser Trp Ala Ala Leu Pro Asn SerGly Gln Gly Gln Lys Glu Gly Val 275 280 285 tgt ggg gca tct cca gag gatgaa gct gaa gag gag gaa gag gag gag 912 Cys Gly Ala Ser Pro Glu Asp GluAla Glu Glu Glu Glu Glu Glu Glu 290 295 300 gag gag gag gaa gag tgt gaaccc cag gca gtg cca gtg tct ccg gcc 960 Glu Glu Glu Glu Glu Cys Glu ProGln Ala Val Pro Val Ser Pro Ala 305 310 315 320 tca gct tgc agc ccc ccgctg cag cag cca cag ggc tcc cgg gtg ctg 1008 Ser Ala Cys Ser Pro Pro LeuGln Gln Pro Gln Gly Ser Arg Val Leu 325 330 335 gcc acc cta cgt ggc caggtg ctc ctg ggc agg ggc gtg ggt gct ata 1056 Ala Thr Leu Arg Gly Gln ValLeu Leu Gly Arg Gly Val Gly Ala Ile 340 345 350 ggt ggg cag tgg tgg cgtcga cgg gcg cag ctg acc cgg gag aag cgc 1104 Gly Gly Gln Trp Trp Arg ArgArg Ala Gln Leu Thr Arg Glu Lys Arg 355 360 365 ttc acc ttc gtg ctg gctgtg gtc att ggc gtt ttt gtg ctc tgc tgg 1152 Phe Thr Phe Val Leu Ala ValVal Ile Gly Val Phe Val Leu Cys Trp 370 375 380 ttc ccc ttc ttc ttc agctac agc ctg ggc gcc atc tgc ccg aag cac 1200 Phe Pro Phe Phe Phe Ser TyrSer Leu Gly Ala Ile Cys Pro Lys His 385 390 395 400 tgc aag gtg ccc catggc ctc ttc cag ttc ttc ttc tgg atc ggc tac 1248 Cys Lys Val Pro His GlyLeu Phe Gln Phe Phe Phe Trp Ile Gly Tyr 405 410 415 tgc aac agc tca ctgaac cct gtt atc tac acc atc ttc aac cag gac 1296 Cys Asn Ser Ser Leu AsnPro Val Ile Tyr Thr Ile Phe Asn Gln Asp 420 425 430 ttc cgc cgt gcc ttccgg agg atc ctg tgc cgc ccg tgg acc cag acg 1344 Phe Arg Arg Ala Phe ArgArg Ile Leu Cys Arg Pro Trp Thr Gln Thr 435 440 445 gcc tgg tga 1353 AlaTrp 450 4 450 PRT Homo sapiens 4 Met Asp His Gln Asp Pro Tyr Ser Val GlnAla Thr Ala Ala Ile Ala 1 5 10 15 Ala Ala Ile Thr Phe Leu Ile Leu PheThr Ile Phe Gly Asn Ala Leu 20 25 30 Val Ile Leu Ala Val Leu Thr Ser ArgSer Leu Arg Ala Pro Gln Asn 35 40 45 Leu Phe Leu Val Ser Leu Ala Ala AlaAsp Ile Leu Val Ala Thr Leu 50 55 60 Ile Ile Pro Phe Ser Leu Ala Asn GluLeu Leu Gly Tyr Trp Tyr Phe 65 70 75 80 Arg Arg Thr Trp Cys Glu Val TyrLeu Ala Leu Asp Val Leu Phe Cys 85 90 95 Thr Ser Ser Ile Val His Leu CysAla Ile Ser Leu Asp Arg Tyr Trp 100 105 110 Ala Val Ser Arg Ala Leu GluTyr Asn Ser Lys Arg Thr Pro Arg Arg 115 120 125 Ile Lys Cys Ile Ile LeuThr Val Trp Leu Ile Ala Ala Val Ile Ser 130 135 140 Leu Pro Pro Leu IleTyr Lys Gly Asp Gln Gly Pro Gln Pro Arg Gly 145 150 155 160 Arg Pro GlnCys Lys Leu Asn Gln Glu Ala Trp Tyr Ile Leu Ala Ser 165 170 175 Ser IleGly Ser Phe Phe Ala Pro Cys Leu Ile Met Ile Leu Val Tyr 180 185 190 LeuArg Ile Tyr Leu Ile Ala Lys Arg Ser Asn Arg Arg Gly Pro Arg 195 200 205Ala Lys Gly Gly Pro Gly Gln Gly Glu Ser Lys Gln Pro Arg Pro Asp 210 215220 His Gly Gly Ala Leu Ala Ser Ala Lys Leu Pro Ala Leu Ala Ser Val 225230 235 240 Ala Ser Ala Arg Glu Val Asn Gly His Ser Lys Ser Thr Gly GluLys 245 250 255 Glu Glu Gly Glu Thr Pro Glu Asp Thr Gly Thr Arg Ala LeuPro Pro 260 265 270 Ser Trp Ala Ala Leu Pro Asn Ser Gly Gln Gly Gln LysGlu Gly Val 275 280 285 Cys Gly Ala Ser Pro Glu Asp Glu Ala Glu Glu GluGlu Glu Glu Glu 290 295 300 Glu Glu Glu Glu Glu Cys Glu Pro Gln Ala ValPro Val Ser Pro Ala 305 310 315 320 Ser Ala Cys Ser Pro Pro Leu Gln GlnPro Gln Gly Ser Arg Val Leu 325 330 335 Ala Thr Leu Arg Gly Gln Val LeuLeu Gly Arg Gly Val Gly Ala Ile 340 345 350 Gly Gly Gln Trp Trp Arg ArgArg Ala Gln Leu Thr Arg Glu Lys Arg 355 360 365 Phe Thr Phe Val Leu AlaVal Val Ile Gly Val Phe Val Leu Cys Trp 370 375 380 Phe Pro Phe Phe PheSer Tyr Ser Leu Gly Ala Ile Cys Pro Lys His 385 390 395 400 Cys Lys ValPro His Gly Leu Phe Gln Phe Phe Phe Trp Ile Gly Tyr 405 410 415 Cys AsnSer Ser Leu Asn Pro Val Ile Tyr Thr Ile Phe Asn Gln Asp 420 425 430 PheArg Arg Ala Phe Arg Arg Ile Leu Cys Arg Pro Trp Thr Gln Thr 435 440 445Ala Trp 450 5 19 DNA Artificial Sequence Description of ArtificialSequence PCR primer pair 5 ggggcgacgc tcttgtcta 19 6 19 DNA ArtificialSequence Description of Artificial Sequence PCR primer pair 6 ggtctccccctcctccttc 19 7 18 DNA Artificial Sequence Description of ArtificialSequence PCR primer pair 7 gcagcaaccg cagaggtc 18 8 19 DNA ArtificialSequence Description of Artificial Sequence PCR primer pair 8 gggcaagaagcagggtgac 19 9 20 DNA Artificial Sequence Description of ArtificialSequence PCR primer pair 9 agggtgtttg tggggcatct 20 10 21 DNA ArtificialSequence Description of Artificial Sequence PCR primer pair 10caagctgagg ccggagacac t 21

1. A DNA sequence comprising a nucleotide sequence encoding a variantα_(2B)-adrenoceptor protein with a deletion of at least 1 glutamate froma glutamic acid repeat element of 12 glutamates, amino acids 298-309, inan acidic stretch of 18 amino acids 294-311, located in the 3^(rd)intracellular loop of the receptor polypeptide.
 2. The DNA sequenceaccording to claim 1 comprising a nucleotide sequence encoding a variantα_(2B)-adrenoceptor protein with a deletion of 3 glutamates, amino acids307-309, from said glutamic acid repeat element of 12 glutamates, aminoacids 298-309, in said acidic stretch of 18 amino acids 294-311, locatedin the 3^(rd) intracellular loop of the receptor polypeptide.
 3. The DNAsequence according to claim 2 comprising the genomic nucleotide sequenceof SEQ ID NO:
 1. 4. The DNA sequence according to claim 1 wherein saidDNA sequence is cDNA.
 5. An RNA sequence comprising an RNA sequencecorresponding to the DNA sequence of claim 1 .
 6. A variantα_(2B)-adrenoceptor protein having a deletion of at least 1 glutamatefrom said glutamic acid repeat element of 12 glutamates, amino acids298-309, in said acidic stretch of 18 amino acids 294-311, located inthe 3^(rd) intracellular loop of the receptor polypeptide.
 7. A variantα_(2B)-adrenoceptor protein according to claim 6 having a deletion of 3glutamates, amino acids 307-309, from said glutamic acid repeat elementof 12 glutamates, amino acids 298-309, in said acidic stretch of 18amino acids 294-311, located in the 3^(rd) intracellular loop of thereceptor polypeptide.
 8. The variant protein according to claim 7comprising the amino acid sequence of SEQ ID NO:
 2. 9. An assay fordetermining the presence or absence of a variant gene as defined inclaim 1 .
 10. The assay according to claim 9 wherein the assay is aDNA-assay.
 11. A method for determining the presence or absence in abiological sample of a DNA sequence as defined in claim 1 , wherein saidDNA, which appears in single stranded form (target nucleic acid), isbrought into contact with a capturing nucleic acid probe and a detectornucleic acid probe, after which the complex “capturing probe-targetnucleic acid-detector probe” is detected.
 12. The method according toclaim 11 , wherein the capturing nucleic acid probe is attached orcapable of attaching to a solid phase, and comprises the cDNA sequenceaccording to claim 4 , wherein a detected signal from the solid phase isan indication of the presence in the sample of a DNA as defined in claim1 .
 13. The method according to claim 11 , wherein the capturing nucleicacid probe is attached or capable of attaching to a solid phase, andcomprises the cDNA corresponding to the gene coding anα_(2B)-adrenoceptor without the deletion defined in claim 1 , wherein adetected signal from the solid phase is an indication of the absence inthe sample of a DNA as defined in claim 1 .
 14. A method for screening asubject to determine if said subject is a carrier of a said variant genewith both alleles encoding a said variant α_(2B)-adrenoceptor, i.e. todetermine if said subject's genotype of the human α_(2B)-adrenoceptor isof the deletion/deletion (D/D) type, comprising the steps of a)providing a biological sample of the subject to be screened, b)providing an assay for detecting in the biological sample the presenceof i) the insertion/insertion (I/I) or deletion/insertion (D/I)genotypes of the human α_(2B)-adrenoceptor, or ii) the D/D genotype ofthe human α_(2B)-adrenoceptor, and c) assessing at least one of the twofollowing i) an individual's risk to develop a disease involvingvascular contraction of coronary arteries, or ii) an individual's needfor α_(2B)-selective or α_(2B)-nonselective α₂-adrenoceptor antagonisttherapy, based on whether said subject is of said D/D genotype or not.15. The method according to claim 14 wherein the assay is a DNA-assay.16. A capturing probe which comprises a single strand of the cDNAaccording to claim 4 .
 17. A capturing probe which comprises a singlestrand of the cDNA corresponding to the α_(2B)-adrenoceptor without thedeletion defined in claim 1
 18. A method for treating a mammal sufferingfrom vascular contraction of coronary arteries, said method comprisingadministering a selective α_(2B)-adrenoceptor antagonist to said mammal.19. The method according to claim 18 wherein said mammal suffers from adisease involving said vascular contraction of coronary arteries. 20.The method according to claim 19 wherein said disease is clinicallyexpressed as coronary heart disease or chronic angina pectoris.
 21. Themethod according to claim 19 wherein said disease is clinicallyexpressed as acute myocardial infarction.
 22. The method according toclaim 20 wherein said chronic angina pectoris is unstable.
 23. Themethod according to claim 20 wherein said chronic angina pectoris isclinically expressed as Prinzmetal's variant form.
 24. A transgenicanimal which carries a human DNA sequence comprising a nucleotidesequence encoding a variant α_(2B)-adrenoceptor protein with a deletionof at least 1 glutamate from a glutamic acid repeat element of 12glutamates, amino acids 298-309, in an acidic stretch of 18 amino acids294-311, located in the 3^(rd) intracellular loop of the receptorpolypeptide.
 25. A transgenic animal according to claim 24 encoding avariant α_(2B)-adrenoceptor protein with a deletion of 3 glutamates,amino acids 307-309.
 26. A transgenic animal which carries a human DNAsequence encoding a α_(2B)-adrenoceptor protein without said deletion ofat least 1 glutamate from a glutamic acid repeat element of 12glutamates, amino acids 298-309, in an acidic stretch of 18 amino acids294-311, located in the 3^(rd) intracellular loop of the receptorpolypeptide.